title: The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO Purification, properties and characterization of mutants.
abstract: The pyruvate dehydrogenase complex of Pseudomonas aeruginosa PAO was purified by affinity chromatography on ethanol-Sepharose 2B followed by sucrose density gradient centrifugation. The overall purification was 130-fold based on enzyme activity. The purified complex contained three major and one minor polypeptide components when analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. These were identified by heat treatment, limited proteolysis and peptide mapping as pyruvate dehydrogenase (El; Mr 92500), acetyltransferases (E2; major component, Mr 76000, and minor component, Mr 77800) and lipoamide dehydrogenase (E3; Mr 58000). The purified complex had a sedimentation coefficient of 48S and the specific activity for the overall reaction of the complex was 6.5 micromol substrate transformed (mg protein)-1 min-1 at the optimum pH (7.8) and 25 degrees C. The lesions in four ace mutants lacking overall pyruvate dehydrogenase complex activity were identified after partial purification of the corresponding cell-free extracts. Three strains, designated ace A mutants, lacked pyruvate dehydrogenase activity (E1 component) and one strain, and ace B mutant, lacked the activity of the acetyltransferase (E2 component).
mesh_terms: D000906:Antibodies / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D005779:Immunodiffusion; D008970:Molecular Weight; D009154:Mutation; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D011768:Pyruvate Dehydrogenase Complex / Q000276:immunology / Q000302:isolation & purification*
chemical_list: D000906:Antibodies; D011768:Pyruvate Dehydrogenase Complex